Schlee et al., Immunity, 2009, 31, 25-34 describe blunt-ended double stranded RNAs carrying a 5′-O-triphosphate moiety on one of the strands that act as potent stimulators of the immune system by binding the RIG-I helicase. Thus, there is a need to provide a simple and efficient method for preparing triphosphate-modified oligonucleotides in high purity, suitable for pharmaceutical applications.
The coupling of triphosphate groups or analogues thereof to the 5′-OH group of nucleosidic compounds is well known in the art. Ludwig J. et al., J. Org. Chem., 1989, 54, 631-635 disclose a solution triphosphorylation method for preparing 5′-O-triphosphates of nucleosides and analogues using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one as the phosphitylating agent. Gaur R. K. et al., 1992, Tetrahedron Letters, 33, 3301-3304 describe the use of said method on solid-phase for the synthesis of 2′-O-methylribonucleoside 5′-O-triphosphates and their Pα-thio analogues. U.S. Pat. No. 6,900,308 B2 discloses the solid-phase synthesis of modified nucleoside 5′-O-triphosphates as potential antiviral compounds and U.S. Pat. Nos. 7,285,658, 7,598,230 and 7,807,653 disclose triphosphate analogues of nucleosides with modifications in the sugar, nucleobase and in the triphosphate entity.
WO96/40159 describes a method for producing capped RNA or RNA analogue molecules, wherein an RNA or RNA analogue oligonucleotide is reacted with a phosphitylating agent such as 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one or a ring-substituted derivative thereof. The resulting intermediate is reacted with a phosphate or pyrophosphate or salt thereof, oxidized or hydrolyzed. The di- or triphosphorylated RNA or RNA analogue is capped by reacting with an activated m7G tri-, di- or monophosphate or analogue.
WO 2009/060281 describes immune stimulatory oligoribonucleotide analogues containing modified oligophosphate moieties and methods for the preparation of such compounds. This method includes the synthesis of the oligonucleotide on a solid support, reacting a nucleotide at a 5′-end of the oligonucleotide with a phosphitylating agent such as 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in a suitable solvent and in the presence of a base, reacting the phosphitylated oligonucleotide with a pyrophosphate or pyrophosphate analogue, oxidizing the oligonucleotide with an oxidizing agent and deprotecting the oligonucleotide to give a triphosphate- or triphosphate analogue-modified oligonucleotide.
Polyacrylamide gel-electrophoresis as employed in WO 96/40159 is applicable only for small scale separations. The resolution power of ion exchange chromatography for 5′-mono-, di-, triphosphorylated products of longer oligoribonucleotides is limited. The required denaturing conditions make separation a tedious task (Sproat, 1999; Zlatev, 2010; WO 2009/060281), moreover, products are usually contaminated with n-1, n-2 sequences and their mono- and diphosphates resulting in insufficient purity. Given the sensitivity for precise terminal structures of the RIG-I ligands, these purification methods are suboptimal for pharmacological applications.
Thus, there is a high need for new triphosphorylated oligonucleotides and analogues thereof, in particular having RIG-I selectivity as well as methods for preparing such compounds.
The present invention therefor relates to novel 5′-triphosphorylated oligonucleotides and analogues thereof which can be produced in large scales for potential clinical use as well as a convenient preparation method for such oligonucleotides. Furthermore, modifications of oligonucleotides are described which establish, maintain and/or improve the RIG-I selectivity of the oligonucleotides or enhance their chemical stability.
Thus, a first aspect of the present invention relates to a modified oligonucleotide of Formula (I)
                wherein V1, V3 and V5 are independently in each case selected from O, S and Se;        V2, V4 and V6 are independently in each case selected from OH, OR1, SH, SR1, F, NH2, NHR1, N(R1)2 and BH3−M+,        W1 is O or S,        W2 is O, S, NH or NR2,        W3 is O, S, NH, NR2, CH2, CHHaI or C(HaI)2,        R1, R2 and R3 are selected from C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 acyl or a cyclic group, each optionally substituted,        or wherein two R1 may form a ring together with an N-atom bound thereto,        M+ is a cation,        X is NH, NR3, O or S,        Z represents a capture tag or H,        Y represents a bond or a linker connecting the capture tag to X, and        ON represents an oligonucleotide comprising at least 4 nucleotide or nucleotide analogue building blocks.        
The term “oligonucleotide” in the context of the present application encompasses compounds comprising a plurality, e.g. at least 4 nucleotide or nucleotide analogue building blocks. Preferably, the oligonucleotide comprises 6-100, e.g. 20-40 building blocks. The nucleotide or nucleotide analogue building blocks may comprise nucleoside or nucleoside analogue subunits connected by inter-subunit linkages. The nucleoside subunits include deoxyribonucleoside subunits, ribonucleoside subunits and/or analogues thereof, particularly sugar- and/or nucleobase-modified nucleoside analogues. Further, the oligonucleotides may comprise non-nucleotidic building blocks and/or further terminal and/or side-chain modifications.
In preferred sugar-modified subunits the 2′-OH of a ribonucleoside subunit is replaced by a group selected from OR, R, halo, SH, SR, NH2, NHR, NR2 or CN, wherein R is C1-6 alkyl, C2-6 alkenyl or C2-6 alkynyl and halo is F, Cl, Br or I. In further preferred sugar-modified subunits, the ribose may be substituted, e.g. by another sugar, for example a pentose such as arabinose. This sugar modification may be combined with 2′-OH modifications as described above, such as in 2′-fluoroarabinonucleoside subunits. Still further preferred sugar-modified subunits include locked nucleosides (LNA) or 2′,3′-seco-nucleosides (UNA). In preferred nucleobase-modified nucleosidic building blocks, a non-standard, e.g. non-naturally occurring nucleobase, is used instead of a standard nucleobase. Examples of non-standard nucleobases are uracils or cytosines modified at the 5-position, e.g. 5-(2-amino)propyl uracil or 5-bromouracil; hypoxanthine; 2,6-diaminopurine; adenines or guanines modified at the 8-position, e.g. 8-bromoguanine; deazanucleosides, e.g. 7-deazaguanine or 7-deazaadenine; or O- and N-alkylated nucleobases, e.g. N6-methyladenine, or N6,N6-dimethyladenine. Further suitable nucleobase analogues may be selected from universal nucleobase analogues such as 5-nitroindole.
The inter-subunit linkage between subunits may be a phosphodiester linkage or a modified linkage, e.g. a phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, boranophosphate, or another modified linkage known to a skilled person in the art.
The oligonucleotide may be selected from deoxyribonucleotides, ribonucleotides and oligonucleotide analogues. The deoxyribonucleotides, ribonucleotides and/or oligonucleotide analogues may be chemically modified at the nucleoside and/or ribose subunit of the analogue, deoxyribonucleotide, ribonucleotide and/or oligonucleotide. Analogues of desoxyribonucleotides or ribonucleotides may comprise at least one desoxyribonucleoside or ribonucleoside subunit and at least one modified nucleosidic subunit and/or at least one modified inter-subunit linkage, e.g. as described above. Oligonucleotide analogues may also consist in their entirety of modified nucleosidic subunits.
The oligonucleotide may be a single-stranded molecule or a double-stranded molecule. Double-stranded oligonucleotides may comprise completely or partially complementary strands. Double-stranded molecules may be blunt-ended or comprise at least one overhang, e.g. a 5′-or 3′-overhang. Overhangs, if present, are preferably located at the distal end of the molecule (with regard to the triphosphate/triphosphate analogue group). Double-stranded oligonucleotides may also comprise a hairpin-structure, wherein the duplex is closed by a loop at the distal end thereof (with regard to the triphosphate/triphosphate analogue group). The loop may comprise nucleotide and/or non-nucleotide building blocks, for example diol-based building blocks such as ethylene glycol moieties, e.g. tri(ethylene)glycol or hexa(ethylene)glycol; propane-1,3-diol; dodecane-1,12-diol; or 3,12-dioxa-7,8-dithiatetradecane-1,14-diol.
In a preferred embodiment, double-stranded molecules are blunt-ended, particularly at the proximal end thereof (with regard to the triphosphate/triphosphate analogue group).
According to an especially preferred embodiment the oligonucleotide is double-stranded, wherein each strand of the double strand has a length of at least 19 nucleotides. A blunt-ended double-stranded oligonucleotide of such a length is especially preferred. According to a further preferred embodiment each strand of the oligonucleotide has a length of at least 19 to 50 nucleotides, 19 to 30 nucleotides, 20 to 30 nucleotides, 22 to 28 nucleotides, especially preferred 22 to 26 nucleotides.
The oligonucleotide may comprise further terminal and/or side-chain modifications, e.g. cell specific targeting entities covalently attached thereto. Those entities may promote cellular or cell-specific uptake and include, for example lipids, vitamins, hormones, peptides, oligosaccharides and analogues thereof. Targeting entities may e.g. be attached to modified nucleobases or non-nucleotidic building blocks by methods known to the skilled person.
According to a preferred embodiment modifications establish and/or enhance the selectivity of the oligonucleotide towards a given target. In a particularly preferred embodiment the RIG-I selectivity of the oligonucleotide is established or enhanced. Methods to determine the RIG-I selectivity of a given oligonucleotide are described herein in detail (cf. Examples) and/or are known to the person skilled in the art.
According to another preferred embodiment the chemical modifications maintain or enhance the chemical stability of the oligonucleotide. A person skilled in the art knows methods for determining the chemical stability of a given oligonucleotide. Such methods are also described, e.g., in the Examples.
According to a preferred embodiment the chemical modifications of the oligonucleotide are independently selected from the group comprising halogenation, in particular F-halogenation, 2′-O-alkylation, in particular 2′-O-methylation, and/or phosphorothioate modifications of internucleotide linkages. Particularly F-halogenation and phosphorothioate modifications increase the stability of the oligonucleotide, while 2′-O-methylation establishes or increases RIG-I selectivity of the oligonucleotide. 2′-O-methylations are also able to modify the immunogenicity of RNA. In a preferred embodiment an oligonucleotide, only comprises one or two 2′-O-methylations per strand, more preferably one 2′-O-methylation per strand.
The 2′-F substitution is particularly preferred. At the 2′ position of the ribose the hydroxyl group is substituted for fluoro. 2′-F substitutions in RNAs particularly result in an enhanced stability against nuclease digestion. In a further embodiment, a 2′-fluoro-substitution may particularly augment a RIG-I-dependent immune stimulation.
Herein, phosphorothioate compounds in general relate to phosphorothioate modifications of internucleotid linkages.
Phosphorothioate-modified compounds having a modification at a terminal end of the oligonucleotide are especially preferred. During phosphorothioate modification the non-binding oxygen atom of the bridging phosphate is substituted for a sulfur atom in the backbone of a nucleic acid. This substitution reduces the cleavability by nucleases at this position significantly and results in a higher stability of the nucleic acid strand.
In an especially preferred embodiment an oligonucleotide according to the present invention shows F-halogenation, methylation, in particular 2′-O-methylation, as well as phosphorothioate modifications, in particular at a terminal end of the oligonucleotide.
The identification patterns of a given oligonucleotide depend on the sequence and the length of an oligonucleotide and can be determined for each given oligonucleotide. A person skilled in the art is well aware how to carry out this determination.
As explained above already, such methods for determining RIG-I selectivity and/or stability of a given oligonucleotide are described in detail in the present application.
The oligonucleotide of formula (I) or (IV) comprises a triphosphate/triphosphate analogue group. In this group, V1, V3 and V5 are independently selected from O, S and Se. Preferably, V1, V3 and V5 are O. V2, V4 and V6 are in each case independently selected from OH, OR1, SH, SR1, F, NH2, NHR1, N(R1)2 and BH3−M+. Preferably, V2, V4 and V6 are OH. R1 may be C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-6 acyl or a cyclic group, e.g. a C3-8 cyclo(hetero)alkyl group, a C3-8 cyclo(hetero)alkenyl group, phenyl or C5-6 heteroaryl group, wherein heteroatoms are selected from N, O and S. Further, two R1 may form a ring, e.g. a 5- or 6-membered ring together with an N-atom bound thereto. R1 may also comprise substituents such as halo, e.g. F, Cl, Br or I, O(halo)C1-2 alkyl and —in the case of cyclic groups—(halo)C1-2 alkyl. M+ may be an inorganic or organic cation, e.g. an alkali metal cation or an ammonium or amine cation.
W1 may be O or S. Preferably, W1 is O. W2 may be O, S, NH or NR2. Preferably, W2 is O. W3 may be O, S, NH, NR2, CH2, CHHaI or C(HaI)2. Preferably, W3 is O, CH2 or CF2. R2 may be selected from groups as described for R1 above. HaI may be F, Cl, Br or I.
According to an especially preferred embodiment V1, V2, V3, V4, V5, V6, W1, W2 and W3 are O.
The triphosphate/triphosphate analogue group is preferably attached to a terminus of the oligonucleotide. Preferably, the group is attached to the 5′-terminus of the oligonucleotide, particularly to the 5′-OH-group of the 5′-terminal sugar thereof.
As defined herein, Z represents a capture tag or H. The capture tag Z can be functionally defined by a series of plausible examples as presented below. A general rule may be: Z has to allow a convenient purification and it should be removable under conditions which are compatible with pppRNA stability requirements. A person skilled in the art is able to determine without undue burden whether a given tag fulfils the functional definition or not. Thus, a person skilled in the art is aware of such capture tags, in particular with regard to the detailed examples given in the present application.
According to a preferred embodiment the capture tag Z is selected from a long-chain aliphatic residue, a partner of a non-covalent high-affinity binding pair, a reactive chemical entirety, Q or NHC2-C24alkyl, Q being preferably selected from H, amino acids, amino acid analogues, C1-C24alkyl, preferably C12-C24alkyl, peptides and lipids. However, according to an especially preferred embodiment Z is decyl, i.e. C10 alkyl.
The capture tag Z according to the present invention is a moiety capable of non-covalently or covalently interacting with a capture reagent under conditions which allow separation for compounds comprising the capture tag, e.g. the oligonucleotide (I) from other species, which do not contain the capture tag. Preferably, the capture reagent is an immobilized reagent or a reagent capable of being immobilized.
Suitable capture tags are for instance long-chain, e.g. C8-24, preferably C13-24, more preferably C13-C14 aliphatic alkyl residues such as octadecyl or other lipidic/lipophilic residues such as e.g. cholesteryl, tocopheryl or trityl and derivatives thereof. However, according to an especially preferred embodiment Z is a decyl residue. In this case, the tagged triphosphate entity can be captured and purified on a solid phase by standard reversed phase chromatography, e.g. RP-HPLC, or by hydrophobic interaction chromatography (HIC). The capture tag may also be a perfluoroalkyl entity, e.g. a 4-(1H,1H,2H,2H-perfluorodecyl)benzyl or a 3-(perfluorooctyl)propyl residue for specific capture of the modified oligo-triphosphate on a Fluorous Affinity support such as is commercially available from Fluorous Technologies, Inc.
In another embodiment, the capture tag may be a first partner of a non-covalent high-affinity binding pair, such as biotin, or a biotin analogue such as desthiobiotin, a hapten or an antigen, which has a high affinity (e.g. binding constant of 10-6 l/mol or less) with the capture reagent, which is a second complementary partner of the high-affinity binding pair, e.g. a streptavidin, an avidin or an antibody.
In yet another embodiment, the capture tag may be a first partner of a covalent binding pair, which may form a covalent bond with the capture reagent, which is a second complementary partner of the covalent binding pair, wherein the covalent bond may be a reversible or an irreversible bond. In this embodiment, the capture tag component Z may be a reactive chemical entity such as an azide or alkynyl group enabling covalent reaction with a capture reagent that contains a complementary reactive group, e.g. an alkynyl or azido moiety, respectively, in the case of the Husigen 3+2 cycloaddition reaction (the so-called “click-reaction” that is Cu(I) catalyzed or a variant thereof that proceeds without Cu(I) ions via release of severe ring strain in e.g. cyclooctyne derivatives). A specific example for Z-Y-X in such a case would be propargylamino.
In another embodiment, the capture tag component may be a chemical entity which contains an additional nucleophilic group, for instance a second amino group in an NH2-Y—XH type reagent. A wide range of suitable electrophilic Z reagent such as cholesterol, chloroformiate or biotin N-hydroxy succinimide active esters may then be used to introduce the tagging group while the oligonucleotide is attached to the solid phase, thus significantly extending the scope of the tagging reaction.
In a preferred embodiment the capture tag is a long-chain alkyl residue, a perfluoroalkyl entity, an azide or an alkynyl group.
In a further embodiment of the present invention, the oligonucleotide may carry a second capture tag at a different position, e.g. at the 3′-terminus. The first and the second capture tags are preferably selected as to allow purification by two orthogonal methods to enable recovery of extremely high purity material. For example the first capture tag may be a lipophilic group, which interacts with a suitable chromatographic support and the second capture tag may be biotin, which interacts with streptavidin.
The second capture tag may be conveniently introduced by performing the synthesis using a modified CPG (controlled glass support) for oligoribonucleotide synthesis.
Y represents a chemical bond or a linker, e.g. an alkylene, preferably a C1-6-alkylene linker, more preferably a C2-5-alkylene linker, or aralkylene linker, optionally comprising heteroatoms or heteroatom-containing groups, such as O, S, NH, C═O or C═S, and/or optionally comprising C═C or C≡C bonds. According to an especially preferred embodiment Y is a bond.
In another preferred embodiment the linker is a polyalkylene oxide, preferably a poly-C2-C6-alkylene oxide, more preferably a poly-C2-C3-alkylene oxide. The number average molecular weight of the linker may be in the range from 30-800 g/mol, preferably from 40-450 g/mol, more preferably from 40-250 g/mol. The linker may be [—CH2CHR4-O-]n with n=1-10, preferably n=1-7, more preferably n=2-5, and even more preferably n=3. R4 may be H or C1-6-alkyl. Further preferred embodiments of Y are shown in FIG. 4. In a preferred embodiment R4 is H.
According to an especially preferred embodiment,    X is NH or O,    Y is —K—((CHR1)m, —CH2—O)n—R—, or    (O—(CHR3)m3—CH2)n1—(O—(CHR2)m2—CH2)n2—(O—(CHR1)m1—CH2)n3—, and    K is O or NH,    m, m1, m2 and m3 is independently 1 to 12, preferably 1 to 8, more preferably 1 to 5, and even more preferably 1 to 3,    n, n1, n2 and n3 is independently 0 to 20, preferably 0 to 10, more preferably 0 to 5, and even more preferably 0 to 3, and    R1, R2 and R3 is independently is H, C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-C6-acyl or a cyclic group, each optionally substituted and    R is C1-6 alkyl, C2-6 alkenyl, C2-6 alkynyl, C2-C6-acyl or a cyclic group, each optionally substituted. Preferably, R is CH2CH2.
According to an especially preferred embodiment with Y being as defined above, R1 and R2 are H, n1 is O and n2 and n3 are 1. Further preferred embodiments can be taken from FIG. 4.
According to another preferred embodiment as Y is being defined above, R1, R2 and R3 are H and n1, n2 and n3 are 1.
According to a preferred embodiment X is NH, K is NH and Y is (CH2CH2O)n with n as being defined above, wherein K is further substituted with cholesterol-C(O)—, trityl or derivatives thereof.
According to an especially preferred embodiment of the oligonucleotide according to Formula (I) X is NH or O, Y is a bond, and Z is C1-C12alkyl or H, preferably C10, Q or NHC2-C24alkyl, wherein Q is selected from H, amino acids, amino acid analogues, C1-C24alkyl, preferably C12-C24alkyl, peptides and lipids, and V1, V2, V3, V4, V5, V6, W1, W2 and W3 are O.
According to a further preferred embodiment of the oligonucleotide of Formula (I) X is NH or O, Y is a bond, and Z is decyl or H, and V1, V2, V3, V4, V5, V6, W1, W2 and W3 are preferably O.
A further aspect of the present invention relates to a pharmaceutical composition comprising a modified oligonucleotide as defined herein.
The pharmaceutical composition according to the invention may further comprise pharmaceutically acceptable carriers, diluents, and/or adjuvants. The term “carrier” when used herein includes carriers, excipients and/or stabilisers that are non-toxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carriers an aqueous pH buffered solutions or liposomes.
Examples of physiologically acceptable carriers include buffers such as phosphate, citrate and other organic acids (however, with regard to the formulation of the present invention, a phosphate buffer is preferred); anti-oxidants including ascorbic acid, low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatine or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose or dextrins, gelating agents such as EDTA, sugar, alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or non-ionic surfactants such as TWEEN, polyethylene or polyethylene glycol. According to an especially preferred embodiment the compound of the invention is dissolved in sterile deionized water.
Such a composition and/or formulation according to the invention can be administered to a subject in need thereof, particularly a human patient, in a sufficient dose for the treatment of the specific conditions by suitable means. For example, the composition and/or formulation according to the invention may be formulated as a pharmaceutical composition together with pharmaceutically acceptable carriers, diluents and/or adjuvants. Therapeutic efficiency and toxicity may be determined according to standard protocols. The pharmaceutical composition may be administered systemically, e.g. intraperitoneally, intramuscularly, or intravenously or locally such as intranasally, subcutaneously, intradermally or intrathecally. The dose of the composition and/or formulation administered will, of course, be dependent on the subject to be treated and on the condition of the subject such as the subject's weight, the subject's age and the type and severity of the disease or injury to be treated, the manner of administration and the judgement of the prescribing physician.
In a preferred embodiment the pharmaceutical composition is administered intradermally. It is especially preferred that the composition is administered intradermally via tattooing, microneedling and/or microneedle patches.
The compound according to the present invention is preferably dissolved and diluted to the desired concentration in sterile, deionized water (purified water) and is then applied on the shaved, ethanol-disinfected skin using a pipetting device, and subsequently tattooed into the skin.
For tattooing, for example, the water-based pharmaceutical composition according to the invention is intradermally injected into the skin, using a (medical) tattoo device fitted with a multi-needle (single use) needle-tip (such as a 9-needle, single-use tip).
The typical tattooing procedure is as follows: After the water-based pharmaceutical composition is pipetted onto the shaved and ethanol cleaned skin, it is introduced into the tattoo machine's multi-needle tip by placing the running needle tip (running at a speed of, for example, 100-120 Hz, in particular at 100 Hz) gently on top of the droplet of water-based pharmaceutical composition. Once the droplet of water-based pharmaceutical composition is completely adsorbed in the running needle tip, and hence resides in between the running needles, the running tip is gently moved back and forth over the skin, by holding the now filled needle tip in an exact 90 degree angle to the skin. Using this method, the water-based pharmaceutical composition is completely tattooed into the skin. For 50-100 μl of water-based pharmaceutical composition this typically takes 10-15 seconds, over a skin area of 2-4 square centimeters. The benefit of this treatment over standard single intradermal bolus injection, is that the water-based pharmaceutical composition is evenly injected over a larger area of skin, and is more evenly and more precisely divided over the target tissue: By using a 9-needle tip at 100 Hz for 10 seconds, this method ensures 9000 evenly dispersed intradermal injections in the treated skin.
Of course, a person skilled in the art may deviate from and adjust the procedure, depending on the patient or part of the body to be treated. The microneedling procedure may be carried out in close analogy to the tattooing procedure. However, with microneedling the tattoo needle-tip is replaced by a microneedling tip, which ensures more superficial intradermal administration. The water-based pharmaceutical composition is in principle pipetted onto the shaved and ethanol cleaned skin and then administered intradermally using the microneedling tip, in analogy to the tattoo procedure. Microneedling does not have necessity to prior adsorption of the pharmaceutical composition in between the microneedling needles.
Additionally, it is envisioned that microneedle patches coated with, or otherwise harbouring, the pharmaceutical composition can be used for transdermal/intradermal delivery. This has the specific advantage that the intradermal delivery of the pharmaceutical composition can be carried out safely by the recipient him-/herself in without the need for a hospital visit and/or medical specialist tattooing/microneedling intervention. This can significantly add flexibility to treatment schemes, allow highly personalized treatment regimens, lower treatment-associated pain, and lower treatment cost. These patches can be constituted of, but not be limited to, dissolving- or non-dissolving microneedle patches for the time-controlled-, sustained- or bolus transdermal delivery of the pharmaceutical composition.
Another aspect of the present invention relates to a method of preparing an oligonucleotide according to any of claims 1-15, comprising the steps    (a) reacting a compound of formula (IIa)
                wherein V1, V3, V5, V4, V6, W1, W2, W3, and ON are as defined above, wherein ON is protected by at least one protection group, with an oxidizing agent to obtain a compound of formula (IIb)        
                wherein V1, V3, V5, V2, V4, V6, W1, W2, W3 and ON are as defined above, wherein ON is protected by at least one protection group,            (b) reacting a compound of formula (IIb) with a capture tag agent of formula (III),Z—Y—XH  (III),            wherein X, Z, and Y are as defined above, wherein X is preferably O, to obtain a reaction product comprising the oligonucleotide of formula (I), and            (c) deprotection of the at least one ON protection group, and    (d) contacting the reaction product of step (c) with a capture reagent capable of interacting with the capture tag, wherein the contacting takes place under conditions which allow separation of the oligonucleotide (I) from other species contained in said reaction product.
An embodiment, wherein X is O, is especially preferred.
During the inventive method the ON oligonucleotide comprises at least one protection group. The use of protective groups according to the present invention aims in particular at protecting the 2′-OH groups ribose subunit of the applied oligonucleotide. A person skilled in the art knows which protection groups are suitable for synthesis, especially a person working in the field of nucleotide synthesis. Protective groups at the 2′-OH position of the ribose subunit of the oligonucleotide are preferred. In a preferred embodiment of the present invention at the 2′-position of the ribose unit fluoride-labile protective groups are used.
Especially preferred are 2′-O-TBDMS or 2′-O-TOM protective groups. In a particularly preferred embodiment the TBDMS protective group is applied.
Particularly during the synthesis of compounds with X═O, which have an enhanced Z—Y—X—PPP binding stability, a broad spectrum of deprotection conditions may lead to the 2′-OH protective groups being cleaved off.
All known deprotection reagents are suitable to cleave off the TBDMS protective group. In particular, the following reagents may be applied:    (a) triethylamine-trihydrofluoride optionally in combination with a polar solvent,    (b) trialklylamine, triethylamine-trihydrofluoride and a polar solvent,    (c) pyridine-HF and other adducts of hydrofluoride of organic nitrogen bases,    (d) ammonium fluoride,    (e) tetra-n-butyl-ammonium fluoride,    (f) tetramethyl-ammonium fluoride, and other tetraalkyl-ammonium fluorides and combinations thereof.
Step (c) is preferably carried out under conditions which do not cause degradation of the triphosphate moiety, e.g. as described in detail below.
Step (a) of the method of the invention comprises the reaction of cyclic P(V)-P(V)-P(III) species of formula (IIa) with an oxidizing agent. The compound of formula (IIa) may be obtained according to standard methods as described by Ludwig et al, 1989, supra and Gaur et al., 1992, supra, namely by reacting the 5′-terminal OH-group of an oligonucleotide with a trifunctional phosphitylating agent, e.g. 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one under suitable conditions, e.g. in the presence of base (pyridine or diisopropylmethylamine) in a suitable solvent such as dioxane or dichloromethane, and subsequent reaction with pyrophosphate (W3═O) or a modified pyrophosphate (W3 is different from O, e.g. CH2, CCl2, NH or CF2). Preferably, a tri-n-butylammonium salt of the pyrophosphate or modified pyrophosphate in DMF is used. The resulting cyclic P(III)-P(V) intermediate (IIa) is then oxidized under anhydrous conditions, e.g. with a peroxide, such as t-butyl hydroperoxide, cumene hydroperoxide, (10-camphorsulfonyl)oxaziridine. Alternatively, phenylacetyldisulfide (V2═S), or borane-diisopropylethylamine complex (V2═BH3) can also be employed respectively, to give the corresponding cyclic 5′-triphosphate/triphosphate analogue of formula (IIb). Reference in this context is also made to WO 96/40159 or WO 2009/060281, the contents of which are herein incorporated by reference.
Reaction step (a) may take place with an oligonucleotide in solution or with an oligonucleotide bound to a solid phase, e.g. an organic resin or glass, such as CPG. The oligonucleotide may further comprise protecting groups, e.g. sugar- or nucleobase protecting groups that are well known to the skilled person. Preferred examples of protecting groups are 2-cyanoethyl for the internucleoside phosphodiester or phosphorothioate, tert-butyldimethylsilyl, triisopropylsilyloxymethyl or bis(acetoxyethoxy)methyl for the ribose 2′-hydroxyl group, 4-t-butylphenoxyacetyl or phenoxyacetyl, acetyl, isobutyryl, benzoyl for the exocyclic amino groups of the nucleobases. More preferably, step (a) is carried out with a solid-phase bound oligonucleotide.
According to step (b) of the method of the invention, compound (IIb) is reacted with a capture tag agent of formula (III)Z—Y—XH  (III)wherein X is a group selected from NH, NR3, O or and X and Y are as defined above. R3 is defined as described above for R1.
In particular, in case for X being O an agent of limited nucleophilicity such as decanol can be used for ring opening (cf. FIG. 1, step 4). Such a step can be carried out at room temperature, e.g. for 48 h, and allows conversion of the cyclotriphosphate to the desired triphosphate γ-ester.
According to step (c) the ON protection groups are cleaved off.
During step (c), for example, the deprotective reagents (a) and (b) specified above may be followed by deprotection condition with X═O over a period of 20 min to 180 min, more preferably 60 min to about 150 min, in particular about 120 min, at 60-70° C., in particular about 65° C. If X═NH, such reactions are excluded due to different binding stability or can only be carried out under significantly worse reaction parameters, e.g. over a period of 40 h at RT.
Step (d) of the method of the present invention comprises contacting the reaction product of step (b), with a capture reagent capable of interacting with the capture tag Z under conditions which allow separation of the capture tag containing oligonucleotide (I) from other species contained in the reaction product. Before step (d), the solid phase bound oligonucleotide (I) is cleaved from the solid phase and deprotected, i.e. the protection groups are partially or completely removed. The capture reagent is preferably immobilized on a suitable support, e.g. a chromatographic support. In order to provide separation of capture tag containing oligonucleotide (I) from non-capture tag-containing species, the reaction products from step (b) are cleaved from a solid phase and deprotected, if necessary, and subjected to a separation procedure, preferably a chromatographic separation procedure based on the interaction of the capture tag Z with the capture reagent. During the separation step, the purity of the oligonucleotide (I), which is generally in the range of 25-70% for the crude material depending upon the length and complexity of the sequence, may be increased to 90%, 91%, 92%, 93%, 94%, 95% or more. For toxicity studies a purity of >85% is desirable, whereas in late stage clinical trials the purity should be in the range of at least 90-95%. Thus, the present invention provides a way to obtain a high purity pppRNA as would be required for human clinical trials.
In step (d), the capture tag and the capture reagent capable of interacting therewith are preferably selected from (i) a hydrophobic or fluorinated group and a chromatographic material with affinity for hydrophobic or fluorinated groups, e.g. a reversed phase material or a fluorous affinity support; (ii) a first partner of a non-covalent high-affinity binding pair and a second complementary partner of a non-covalent high-affinity binding pair, (iii) a first partner of a covalent binding pair and a second complementary partner of a covalent binding pair, where the first and second partner form covalent bonds.
The capture tag is functionally defined below by a series of plausible Examples. A general rule may be:
Z has to allow a convenient purification, and it should be removable under conditions which are compatible with pppRNA stability requirements.
Additionally, the method may further comprises step (e) removing the capture tag to obtain an oligonucleotide of Formula (IV). According to a preferred embodiment, for X═O a compound of Formula (IV)a or (IV)b
and for X═NH a compound of Formula (IV)a
is obtained.
Step (e) has to be compatible with stability requirements of the triphosphate end product and with stability requirements of the interribonucleotide bond. It may comprise cleavage by mildly acidic conditions when X is NH, cleavage with silver ions when X is S, cleavage by a thiol such as dithiothreitol leading to elimination of thiirane when Y—X—P contains —S—S—CH2—CH2—O—P.
In further embodiments the capture tag Z is not or not completely removed. In these embodiments the tagged oligonucleotide as such may have utility, e.g. utility as pharmaceutical agent.
In these embodiments, the reagent Z—Y—XH has to be selected from a subgroup of Z-residues, which are functionally compatible with the structural requirements of the RIG-I sensor. For instance, the Z=decyl-octadecyl, Y=link X═NH or O combination is known to fulfill these requirements.
The triphosphate/triphosphate analogue modified oligonucleotides produced according to the present invention are particularly suitable for pharmaceutical applications due to their high purity. In an especially preferred embodiment, the oligonucleotide (I) or (IV) is an activator of RIG-I helicase. Specific examples of suitable RIG-I activators are disclosed in Schlee et al., 2009, supra, the content of which is herein incorporated by reference.